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During embryo development, extracellular matrix (ECM) remodeling by matrix metalloproteinases (MMPs) and promotes downstream cell specifications. Pluripotent stem cell (PSC) aggregates can recapitulate various aspects of embryogenesis in vitro, and incorporation of biomaterial microparticles also provides an ideal platform to study cell-biomaterial interactions. Stem cell interactions with ECM-based biomaterials can impact tissue remodeling and differentiation propensity via modulation of MMP activity. This work investigated the MMP activity and subsequent mesenchymal differentiation of embryonic stem cell (ESC) aggregates with incorporated gelatin methacrylate (GMA) MPs with either low (20%) or high (90%) cross-linking densities, corresponding to faster or slower degradation rate, respectively. GMA MP incorporation increased total MMP and MMP-2 levels within 3D ESC aggregates in a substrate-dependent manner. GMA MP-incorporated aggregates also expressed higher levels of epithelial-to-mesenchymal transition markers and displayed enhanced mesenchymal morphogenesis than aggregates without MPs, and the MP-mediated effects were completely abrogated with MMP inhibitor treatment. This work predicts that control of proteolytic responses via introducing ECM-based MPs may offer a novel avenue to engineer the ECM microenvironment to modulate stem cell differentiation.

This thesis is an in-depth case study of an interdisciplinary, paradigm breaking, research team who are seeking to develop cultured red blood cells (RBCs) for transfusion using stem cells (known as the BloodPharma project). It answers the research question: What can an in-depth case study of the BloodPharma project reveal about everyday scientific practice and the project management of a large research programme? The BloodPharma project occupies a unique position within the stem cell arena due to the size and multi-disciplinary nature of the project team, and the unique risk profile of cultured RBCs. The historical significance of blood donation is combined with the modern innovation of stem cell usage, to create a product which is both novel but also highly emotive. The case study comprises interviews with a range of stakeholders, laboratory observation, and participant observation of public outreach activities. In addition presence at team meetings and teleconferences has allowed an in-depth analysis of the project progression. The thesis has also drawn heavily on science and technology studies and scientific literature, as well as on information gathered from a wide variety of conferences and workshops. Key findings indicate that early stage laboratory work in this interdisciplinary project is achieved through the standardisation of work across different research spaces, with training and visual aids used to overcome the hurdle of tacit knowledge associated with the development of stem cell technologies. In designing early stage laboratory work the team looked to the human body as a benchmark of in vivo RBC production, using in vivo cells as a dual standard for which the team must aim, but cannot fall short of. Scale-up and standardisation were identified as the key challenges to the translation of this early stage laboratory work into a clinically useable product. These challenges require new expertise and innovation, and are an example of the translational obstacles of tacit knowledge and visual techniques which are found in the wider stem cell field. The use of target markets was identified by the team as a stepping stone to larger scale production, although in common with other stem cell therapies the clinical trials route to first-in-human use is still unclear. The uncertainty of regulation for stem cell products, and specifically how this relates to the BloodPharma project, is also a key finding of this thesis. Interactions with the regulatory system are seen as a necessity but also represent an area of confusion for laboratory researchers, requiring much specialist knowledge to understand and navigate regulatory documents. Regulatory expertise is brought to the BloodPharma project through reliance on particular members of staff. Public outreach has formed an important part of the BloodPharma project and shows the scientists stepping outside their primary area of expertise, a reflection of the broader trend amongst academic research to demonstrate ‘broader impact criteria’. Public outreach for the BloodPharma team was found to occupy a unique niche, given that the team must balance the promotion of a future product with the preservation of the current donation system. This research is of a case study which goes beyond the boundaries of the laboratory, to look not only at early stage laboratory work, but also at the way in which the team envisions future translation and regulatory hurdles, and the public outreach which must combine to develop a novel stem cell therapy. The thesis is the first in-depth case study to follow a large, interdisciplinary, stem cell team through the work they carry out both within the laboratory space, and outside it; challenging the idea of what it means to carry out scientific work in this novel area of stem cell therapies.

Thesis (PhD)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: HISTORICAL PERSPECTIVE Stem cell therapy was commenced after using rabbits as research models. Once this process was successful, the first human transplant was done in 1974. Certain prerequisites were necessary and these were achieved - a protected environment, an apheresis unit, protocols and accreditation with International Registries. Initially, unmanipulated bone marrow and peripheral blood stem cells were used together with immunosuppressive drugs followed by the use of Cyclosporin A then the addition of ex vivo Campath®. AUDIT OF ACUTE ASSOCIATIONS (468 subjects in initial cohort) NEPHROLOGY Creatinine was used as an indication of renal function. Of the 76 available for analysis, 47% had acute kidney injury. Dialysis had a poor outcome as reported in the literature. Renal complications occurred frequently mostly due to infection. CARDIOLOGY A total of 119 individuals were available for analysis. Echocardiograms and electrocardiograms were part of pre-transplant assessment. Left ventricular systolic dysfunction predicted for increasing post transplant problems. Cardiac complications occurred at a lower frequency than other post-transplants side-effects consistent with the published data. DERMATOLOGY Cases were evaluated on a daily basis and referred to a dermatologist when necessary. To confirm Graft-Versus-Host Disease (GVHD), a skin biopsy was done to differentiate it from drug hypersensitivity or viral infections. The exposure to ex vivo Campath® significantly improved outcome by reducing the incidence and severity of GVHD. Quality of life was enhanced with substantial cost saving. GASTROENTEROLOGY Foregut symptoms occurred in 90% of patients. Nutritional problems were encountered. Altered liver functions were relatively common attributable to drugs, sepsis and conditioning regimens. Liver biopsies were not performed in this series and endoscopy performed only when necessary. A STUDY ON LATE COMPLICATIONS (55 subjects) RESPIRATORY Spirometry and diffusing capacity were done in this cohort. All the lung function studies were within the predicted normal range apart from some marginal reduction in diffusing capacity. In none of these patients did late consequences such as Bronchiolitis Obliterans Organising Pneumonia and Late Onset Non-Infectious Pulmonary complications occur. Cytomegalovirus reactivation was common but early intervention prevented serious complications. IMMUNOLOGY An in vitro functional study was done. Both the innate and adaptive systems were evaluated. Taken into consideration were the type of transplant, age from transplant, diagnosis and conditioning. The granulocyte Burst-test was done for the innate profile. Reduced activity was shown in all the subgroups. It appears as if the innate response of the granulocytic cells never recovered due to reduced granulocytic function in vitro. The adaptive responses were evaluated in vitro and only the autografts showed better CD4+ and CD8+ cytokine production. No major differences were seen in other groups. Normal cytokine production by CD4+ and CD8+ T cells were present when these were activated in vitro to produce regulatory cytokines, implying that their lymphoid component was intact post-transplant. BONE DISEASE Here both the Dual energy X-ray Absorptiometry (DXA) and Quantitative Computed Tomography (QCT) were used to evaluate bone mineral density. There was a discrepancy present between the two modalities. DXA showed no osteoporosis but QCT 22%. Biomarkers were normal in all. There was no history of fracture and no objective evidence of vertebral fractures using vertebral fracture assessment. Although QCT was used for the study, DXA remains the gold standard in South Africa. CONCLUSION This doctoral provided information on the non-haematological consequences in South Africa with the use of Campath® ex vivo. / AFRIKAANSE OPSOMMING: HISTORIESE PERSPEKTIEF Stamsel terapie is voortgesit nadat konyne aanvanklik as navorsingsmodelle gebruik is. Na suksesvolle voltooiing van hierdie proses, is die eerste menslike oorplanting gedoen in 1974. Sekere voorvereistes was nodig en hierdie was bereik – ʼn beskermde omgewing, ʼn aferese eenheid, protokolle en akkreditasie by Internasionale Registers. Aanvanklik is ongemanipuleerde beenmurg- en perifere bloed stamselle gebruik, tesame met immuunonderdrukkende middels, gevolg deur die gebruik van Sikloporien A en daarna die toevoeging van ex vivo Campath®. OUDIT VAN AKUTE ASSOSIASIES (468 GEVALLE IN DIE OORSPRONKLIKE GROEP) NEFROLOGIE Kreatinien is gebruik as ʼn aanduiding van nierfunksie. Van die 76 gevalle beskikbaar vir ontleding, het 47% akute nierbeserings gehad. Dialise het ʼn swak uitkoms gehad soos gerapporteer in publikasies. Nier komplikasies het gereeld voorgekom, meestal as gevolg van infeksie. KARDIOLOGIE ʼn Totaal van 119 gevalle was beskikbaar vir ontleding. Eggokardiogramme en elektrokardiogramme was deel van die pre-oorplanting assessering. Linker ventrikulêre disfunksie was voorspelbaar van verhoogde postoorplanting probleme. Kardiale komplikasies het konstant volgens publikasies minder geredelik voorgekom as ander post-oorplantings newe-effekte. DERMATOLOGIE Gevalle is op ʼn daaglikse basis geëvalueer en verwys na ʼn dermatoloog wanneer nodig. ʼn Velbiopsie is gedoen om “Graft-Versus-Host” siekte (GVHD) te bevestig en dit te onderskei van middel hipersensitiwiteit of virale infeksies. Die blootstelling aan ex vivo Campath® het uitkomste aansienlik verbeter deur die voorkoms en erns van GVHD te verminder. Kwaliteit van lewe is verhoog met aansienlike koste besparing. GASTROENTEROLOGIE Boonste gastro-intestinale simptome het voorgekom in 90% van die pasiënte. Wanvoeding het voorgekom.. Abnormale lewerfunksies was relatief algemeen toeskryfbaar aan middels, sepsis en kondisionerings protokolle. Lewer biopsies is nie in hierdie reeks uitgevoer nie en endoskopie slegs wanneer dit noodsaaklik was. DIE STUDIE VAN LAAT KOMPLIKASIES (55 GEVALLE) RESPIRATORIES Spirometrie en diffusie kapasiteit is gedoen in hierdie groep. Al die longfunksie ondersoeke was binne die voorspelde normale waardes behalwe ʼn paar marginale afnames in die diffusie kapasitiet. In geen van hierdie pasiënte het laat nagevolge soos Bronchoilitis Obliterans Organiserende Pneumonie en Laat Aanvangs Nieinfektiewe Long komplikasies voorgekom nie. Sitomegaal virus heraktivering was algemeen maar vroeë intervensie het ernstige komplikasies voorkom. IMMUNOLOGIE ʼn In vitro funksionele studie is gedoen. Beide die spesifieke en nie-spesifieke immuun stelsels is geëvalueer. Die tipe oorplanting, tyd vanaf oorplanting, diagnose en kondisionering is in ag geneem. Die “Granulocyte Burst” toets is gedoen vir die nie-spesifieke profiel. Verminderde aktiwiteite is bewys in al die subgroepe. Dit wil voorkom asof die nie-spesifieke respons van die granulosiete nooit herstel nie as gevolg van die verlaagde in vitro granulosiet funksie. Die spesifieke immuun respons is in vitro geëvalueer en slegs die outotransplantaat het beter CD4+ en CD8+ sitokiene produksie getoon. Geen groot verskille is gesien in ander groepe nie. By CD4+ en CD8+ T selle was normale sitokiene produksie teenwoordig toe dit in vitro geaktiveer is om regulatoriese sitokiene produksie te produseer, wat beteken dat hul limfoïede komponent na oorplanting ongeskonde was. BEEN SIEKTE Beide die Dubbele energie x-straal absorpsiemetrie (DXA) en Kwantitatiewe rekenaar tomografie (QCT) is hier gebruik om been mineraal digtheid te evalueer. Daar was ʼn teenstrydigheid teenwoordig tussen die twee modaliteite. DXA het geen osteoporose getoon nie maar QCT het 22% getoon. Biomerkers was normaal in albei. Daar was geen geskiedenis van frakture en geen objektiewe bewyse van vertebrale frakture met Vertebrale Fraktuur Assessering nie. Alhoewel QCT gebruik is vir die studie bly DXA die goue standaard in Suid-Afrika. GEVOLGTREKKING Hierdie doktoraal verskaf inligting oor die nie-hematologiese gevolge in Suid-Afrika met die gebruik van Campath® ex vivo.

published_or_final_version / abstract / Orthopaedics and Traumatology / Master / Master of Philosophy

published_or_final_version / abstract / Mechanical Engineering / Master / Master of Philosophy

published_or_final_version / abstract / Medicine / Master / Doctor of Medicine

published_or_final_version / abstract / Medicine / Master / Master of Philosophy

published_or_final_version / Mechanical Engineering / Master / Master of Philosophy